Method for culturing in-vitro embryos of oocyte of living sheep

Abstract

The invention relates to the technical field of animal breeding, and discloses a living sheep oocyte in-vitro embryo culture method, which comprises the following steps: step 1, living body oocyte (LOPU) optimization, step 2, in-vitro maturation (IVM) system optimization, and step 3, embryo culture and quality evaluation. Compared with the prior art, the method has the advantages that the availability rate of oocytes is increased to 67.01%; the blastocyst cell apoptosis rate is reduced to 1.85% (4.95% of a control group); and the number of cells in the cell mass is obviously increased (33.33 vs14.25).

Description

Technical Field

[0001] The invention relates to the technical field of animal reproduction, in particular to a method for in vitro embryo culture of oocytes collected from living sheep. Background Art

[0002] Existing goat in vitro embryo production (IVEP) technology suffers from low oocyte recovery rates (40-60%), asynchronous nuclear and cytoplasmic maturation, and severe oxidative stress damage, resulting in a blastocyst rate of less than 30%. The traditional method of multiple follicle-stimulating hormone (FSH) injections is cumbersome, and the lineage of oocytes derived from slaughterhouses is unknown. Summary of the Invention

[0003] (1) Technical problems solved

[0004] The purpose of the present invention is to solve the problems mentioned in the background technology by optimizing the live oocyte retrieval (LOPU) pressure, the follicle-stimulating hormone (FSH) slow-release technology and the addition of bone morphogenetic protein 15 (BMP15) and flavonoid compound (Eupatilin), and to provide a method for in vitro embryo culture of live oocytes from sheep.

[0005] (2) Technical solution

[0006] To solve the above technical problems, the present invention provides a technical solution: a method for in vitro embryo culture of oocytes collected from living sheep, comprising the following steps:

[0007] Step 1: Live Oocyte Purification (LOPU) Optimization:

[0008] Before egg collection, the donor sheep are given hormone treatment, including:

[0009] Treatment for estrus synchronization: Place progesterone vaginal suppository for 14 days and inject 1 mL of sodium chlorpromazine when removing the suppository;

[0010] Follicle-stimulating hormone (FSH) treatment: A total dose of 150-240 IU is administered as a single injection of hyaluronic acid (HA) sustained-release agent (10 mg / mL) instead of the traditional taper method;

[0011] Use a negative pressure pump to collect eggs. The egg collection equipment uses a 20G needle tip egg collection needle, connected to a negative pressure pump to aspirate follicular fluid. The negative pressure is set to 60-80 mmHg.

[0012] Step 2: In vitro maturation (IVM) system optimization:

[0013] The following components were added to the in vitro maturation (IVM) medium:

[0014] Bone morphogenetic protein 15 (BMP15), concentration: 25-200 ng / mL; flavonoid compound (Eupatilin), concentration: 0.01-2 μM; culture conditions: 38.5°C, 5% CO2 environment, culture time: 22-24 hours;

[0015] Step 3: Embryo culture and quality assessment:

[0016] Mature oocytes were parthenogenetically activated (PA) and cultured in G1 medium until the blastocyst stage. Embryo quality was assessed by blastocyst cell number, inner cell mass / trophoblast ratio, and cell apoptosis rate.

[0017] The concentration of hyaluronic acid (HA) sustained-release agent was 10 mg / mL, mixed with follicle-stimulating hormone (FSH) and injected once to simplify the hormone treatment process. When bone morphogenetic protein 15 (BMP15) and flavonoid compound (Eupatilin) ​​were added together, the blastocyst rate was significantly higher than that of the control group (P<0.05), and the blastocyst apoptosis rate was reduced to 1.85±0.41%. The suction flow rate of the negative pressure pump was 3 mL / min, and the oocyte recovery rate was ≥84.35%.

[0018] As a modification, follicle stimulating hormone (FSH) treatment in step 1: total dose is 210 IU.

[0019] As an improvement, the negative pressure in step 1 is set to 70 mmHg.

[0020] As an improvement, the concentration of bone morphogenetic protein 15 (BMP15) in step 2 is 50 ng / mL.

[0021] As an improvement, the concentration of the flavonoid compound (Eupatilin) ​​in step 2 is 1 μM.

[0022] (3) Beneficial effects

[0023] After adopting the above method, the present invention has the following advantages: the availability rate of oocytes is increased to 67.01%; the apoptosis rate of blastocyst cells is reduced to 1.85% (4.95% in the control group); and the number of cells in the inner cell mass is significantly increased (33.33 vs 14.25). BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the effect of different concentrations of BMP15 on the rate of oocyte first polar body extrusion.

[0025] Figure 2 The effect of different concentrations of BMP15 on the cleavage rate of early PA embryos.

[0026] Figure 3The effect of different concentrations of BMP15 on the blastocyst rate of early PA embryos.

[0027] Figure 4 Representative fluorescence images of mitochondrial distribution types in oocytes.

[0028] Figure 5 The effect of BMP15 on the activity and distribution of mitochondria in oocytes.

[0029] Figure 6 Representative fluorescence images of the distribution types of cortical granules in oocytes.

[0030] Figure 7 The effect of BMP15 on the distribution of granules in the oocyte cortex.

[0031] Figure 8 It is the effect of BMP15 on cumulus cell expansion.

[0032] Figure 9 The effect of BMP15 on cumulus cell apoptosis.

[0033] Figure 10 This is the effect of different concentrations of Eupatilin on the rate of first polar body extrusion of oocytes.

[0034] Figure 11 The effect of different concentrations of Eupatilin on the cleavage rate of early PA embryos.

[0035] Figure 12 The effect of different concentrations of Eupatilin on the blastocyst rate of early PA embryos.

[0036] Figure 13 The effect of Eupatilin on the expansion index of cumulus cells.

[0037] Figure 14 The effect of Eupatilin on oocyte mitochondrial activity.

[0038] Figure 15 The effect of Eupatilin on the mitochondrial membrane potential of oocytes.

[0039] Figure 16 The effect of Eupatilin on the ATP content of oocytes. DETAILED DESCRIPTION

[0040] The following describes embodiments of the present invention in detail, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and are not to be construed as limiting the present invention.

[0041] Example 1

[0042] Combined with attachment Figure 1-16 A method for in vitro embryo culture of live oocytes collected from sheep, comprising the following steps:

[0043] Step 1: Live Oocyte Purification (LOPU) Optimization:

[0044] Before egg collection, the donor sheep are given hormone treatment, including:

[0045] Treatment for estrus synchronization: Place progesterone vaginal suppository for 14 days and inject 1 mL of sodium chlorpromazine when removing the suppository;

[0046] Follicle-stimulating hormone (FSH) treatment: a total dose of 210 IU, administered as a single injection of hyaluronic acid (HA) sustained-release agent (10 mg / mL) instead of the traditional taper method;

[0047] Use a negative pressure pump to collect eggs. The egg collection equipment uses a 20G needle tip egg collection needle, connected to a negative pressure pump to aspirate follicular fluid, and the negative pressure is set to 70mmHg;

[0048] Step 2: In vitro maturation (IVM) system optimization:

[0049] The following components were added to the in vitro maturation (IVM) medium:

[0050] Bone morphogenetic protein 15 (BMP15) at a concentration of 50 ng / mL; flavonoid compound (Eupatilin) ​​at a concentration of 1 μM; culture conditions: 38.5°C, 5% CO2 environment, culture time 22-24 hours;

[0051] Step 3: Embryo culture and quality assessment:

[0052] Mature oocytes were parthenogenetically activated (PA) and cultured in G1 medium until the blastocyst stage. Embryo quality was assessed by blastocyst cell number, inner cell mass / trophoblast ratio, and cell apoptosis rate.

[0053] The concentration of hyaluronic acid (HA) sustained-release agent was 10 mg / mL, mixed with follicle-stimulating hormone (FSH) and injected once to simplify the hormone treatment process. When bone morphogenetic protein 15 (BMP15) and flavonoid compound (Eupatilin) ​​were added together, the blastocyst rate was significantly higher than that of the control group (P<0.05), and the blastocyst apoptosis rate was reduced to 1.85±0.41%. The suction flow rate of the negative pressure pump was 3 mL / min, and the oocyte recovery rate was ≥84.35%.

[0054] Example 2

[0055] Combined with attachment Figure 1-16In the live oocyte collection operation, the donor sheep were estrus synchronized and then injected intramuscularly with 210 IU follicle-stimulating hormone (FSH) (hyaluronic acid (HA) slow-release agent (10 mg / mL)).

[0056] During laparoscopic egg collection, the vacuum pump is set to 70 mmHg and a 20G needle is used to aspirate 2-5 mm follicles;

[0057] After oocyte grading, AC grade COCs were used for subsequent culture.

[0058] Example 3

[0059] Combined with attachment Figure 1-16 In vitro maturation culture, in vitro maturation (IVM) culture medium preparation: TCM199 basal medium supplemented with 50 ng / mL bone morphogenetic protein 15 and 1 μM flavonoid compound (Eupatilin);

[0060] After culturing for 22 h, COCs were parthenogenetically activated (PA) and transferred to G1 culture medium;

[0061] The development rate was calculated at the blastocyst stage, and the cell number and apoptosis rate were evaluated by differential staining.

[0062] Experimental data verification:

[0063] Live Oocyte Purification (LOPU) efficiency: The recovery rate was 84.35% at a negative pressure of 70 mmHg, significantly higher than that of the syringe (40.40%).

[0064] In vitro maturation (IVM) effect: the blastocyst rate in the bone morphogenetic protein 15 (BMP15) group was 29.83% (13.73% in the control group), and the blastocyst rate in the flavonoid compound (Eupatilin) ​​group was 30.77%;

[0065] Embryo quality: The number of cells in the cell clusters of the combined supplementation group was 33.33±3.18, and the apoptosis rate was 1.85±0.41%.

[0066] Although the embodiments of the present invention have been shown and described above, it is understandable that the above embodiments are illustrative and cannot be understood as limiting the present invention. Those skilled in the art may change, modify, replace, modify, delete some features, add features, or re-combine features to form a technical solution within the scope of the present invention without departing from the principles and purpose of the present invention. Any simple modifications, equivalent changes, and modifications made to the above embodiments based on the innovative principles of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for in vitro embryo culture of oocytes collected from living sheep, characterized by: It includes the following steps: Step 1: Live Oocyte Purification (LOPU) Optimization: Before egg collection, the donor sheep are given hormone treatment, including: Treatment for estrus synchronization: Place progesterone vaginal suppository for 14 days and inject 1 mL of sodium chlorpromazine when removing the suppository; Follicle-stimulating hormone (FSH) treatment: A total dose of 150-240 IU is administered as a single injection of hyaluronic acid (HA) sustained-release agent (10 mg / mL) instead of the traditional taper method; Use a negative pressure pump to collect eggs. The egg collection equipment uses a 20G needle tip egg collection needle, connected to a negative pressure pump to aspirate follicular fluid. The negative pressure is set to 60-80 mmHg. Step 2: In vitro maturation (IVM) system optimization: The following components were added to the in vitro maturation (IVM) medium: Bone morphogenetic protein 15 (BMP15), concentration: 25-200 ng / mL; flavonoid compound (Eupatilin), concentration: 0.01-2 μM; culture conditions: 38.5°C, 5% CO2 environment, culture time: 22-24 hours; Step 3: Embryo culture and quality assessment: Mature oocytes were parthenogenetically activated (PA) and cultured in G1 medium until the blastocyst stage. Embryo quality was assessed by blastocyst cell number, inner cell mass / trophoblast ratio, and cell apoptosis rate. The concentration of hyaluronic acid (HA) sustained-release agent was 10 mg / mL, mixed with follicle-stimulating hormone (FSH) and injected once to simplify the hormone treatment process. When bone morphogenetic protein 15 (BMP15) and flavonoid compound (Eupatilin) ​​were added together, the blastocyst rate was significantly higher than that of the control group (P<0.05), and the blastocyst apoptosis rate was reduced to 1.85±0.41%. The suction flow rate of the negative pressure pump was 3 mL / min, and the oocyte recovery rate was ≥84.35%.

2. The method for in vitro embryo culture of oocytes collected from living sheep according to claim 1, characterized in that: Follicle-stimulating hormone (FSH) treatment in step 1: total dose is 210 IU.

3. The method for in vitro embryo culture of oocytes collected from living sheep according to claim 1, characterized in that: The negative pressure in step 1 was set to 70 mmHg.

4. The method for in vitro embryo culture of oocytes collected from living sheep according to claim 1, characterized in that: The concentration of bone morphogenetic protein 15 (BMP15) in step 2 is 50 ng / mL.

5. The method for in vitro embryo culture of oocytes collected from living sheep according to claim 1, characterized in that: The concentration of the flavonoid compound (Eupatilin) ​​in step 2 is 1 μM.

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